Objectives During this reporting period we focused primarily on two technicalissues that are of importance to our project first, the enhancement of the analytical sensitivity of our PCR procedure and second, the improvement of the fixation protocol for brain tissue. Results A search was made for more sensitive procedures and a nested PCR protocol was identified which appeared to have all of the advantages of such protocols vis vis sensitivity without any of its shortcomings with respect to risk of contaminations. The procedure is based on an initial amplification with primers of high GC content, which can be annealed at 70'C. Hence, the "first amplification" can proceed in the presence of the pair of primers that is used in the second (nested) amplification. The latter pair has an annealing temperature of 55'C and is therefore inactive during the first round of amplification. Since all of the primers are present from the beginning, the tubes do not have to be opened mid way. Isopsoralen is included and the amplicon is "sterilized" by exposure to UV light before opening the tube to remove the DNA. The procedure was adapted to the amplification of B. burgdorferi DNA that is contained in cerebral-spinal fluid (CSF), and it was demonstrated that the analytical sensitivity of the procedure permitted the detection of less than one spirochete in CSF. Longitudinally collected samples of CSF from several animals were assessed with the new procedure. All of these same samples had been negative with our previous PCR protocol despite the fact that many of these animals had shown CSF pleocytosis and signs of neuroborreliosis of the central nervous system (CNS) as assessed by somatosensory evoked potential measurements. We have now been able to firmly document invasion of the CNS of rhesus macaques by B. burgdorferi of the NT1 strain. In search for an improved fixation protocol for brain tissue, five macaques were subjected to whole body perfusion and held in the respective fixative for 48 or 96 hours postperfusion. The following fixatives were used (1) 10% buffered formaldehyde; (2) Streck Tissue (zinc Based); (3) Prefer Immunofixative (Dialdehyde Based); (4) Z-Fix (buffered zinc formaldehyde); (5) 1 % zinc formaldehyde (unbuffered). Buffered formaldehyde produced overall good fixation with minimal artifacts in the brain tissue at both 48 and 96 hours post-perfusion. Marginal to poor enolase immunostains were observed. This is the result expected with this fixative, as it does not preserve well the antigenicity of the tissue proteins. However, morphology is very well maintained. The Streck tissue fixative produced marginal fixation at 48 hours and good fixation at 96 hours. The H&E staining produced a well-balanced stained section of these samples. Excessive vacuolation was present at 48 hours and minimal vacuolation at 96 hours post-perfusion. Glial elements were well immunostained. The neuronal immunostain was of marginal quality at 48 hours but acceptable at 96 hours. Prefer and Z-Fix immunofixatives produced good fixation of the brain tissue at both 48 and 96 hours post-perfusion. The H&E staining balance was excellent as well as the glial and enolase immunostains. We feel that we have identified optimal fixation procedures that will permit a thorough analysis of brain tissues postmortem. Future directions Armed as we are now with improved methods to detect spirochetal invasion of the CNS and the damage that such invasion may cause in the brain, we will be able not only to complete this aspect of our study of pathogenesis of Lyme neuroborreliosis, one of our long-term goals, but also to tackle more effectively the antibiotic efficacy trial in which we are also involved.